As recently reviewed, SERT-deficient Slc6a4 +/- and -/- mice have gene-proportionate increases in extracellular fluid serotonin (5-HT) concentrations. i.e., 3- or 6-fold excesses respectively over +/+ mice (Murphy et al., 2008;Murphy &Lesch 2008;Fox et al., 2007). At the same time, the Slc6a4 -/- mice have a marked deficit of intracellular, releasable 5-HT. These mice also have significantly increased 5-HT synthesis and turnover across all five brain regions investigated in Slc6a4 -/- mice, and this increase is significantly greater in female Slc6a4 -/- mice. Expression of the organic cation transporters, OCT1 and OCT3, which are low affinity transporters of monoamines, is increased--indicative of some partial but inefficient attempts at compensation via heterologous transporters. Slc6a4 +/- mice manifest decreased 5-HT clearance and elevated extracellular fluid 5-HT but nonetheless have unchanged tissue 5-HT concentrations in the brain and periphery and unchanged brain 5-HT synthesis and turnover. Thus, a single copy of Slc6a4 is adequate to maintain overall 5-HT tissue homeostasis. The resulting data are: (1). Continuing advances have been made in our studies of the serotonin syndrome. Most commonly, this syndrome occurs as a side effect in humans treated with certain antidepressant and anti-anxiety drugs. Importantly, its occurrence contributes to reduced therapeutic efficacy or a requirement to interrupt treatment in some individuals. Our earlier studies exploring this behavioral and temperature-related syndrome in SERT-deficient mice had discovered that genetic vulnerability to a markedly exaggerated serotonin syndrome was present when these mice were exposed to the metabolic precursor of serotonin, 5-HTP, or to other serotonergic drugs such as the monoamine oxidase inhibiting (MAOI) antidepressant, tranylcypromine. Tranylcypromine is a clinically available antidepressant drug, and 5-HTP is readily available on more than 100 internet sites and sold over-the-counter as a dietary supplement. Spontaneous mild serotonin-syndrome behaviors were also observed in untreated Slc6a4 -/- mice (Fox et al., 2007;Fox et al., 2008). To better define the mechanisms underlying the serotonin syndrome, brain levels of serotonin and its metabolites were measured. In parallel with the greater behavioral changes, SERT -/- mice had 5 to 12-fold increases in serotonin, with smaller 2 to 5-fold increases in SERT +/+ and +/- mice following 5-HTP treatment (Fox et al., 2008). Exaggerated changes in temperature were also found in SERT -/- mice. Different serotonin receptor mechanisms were discovered to be involved with temperature responses by studies with different selective serotonin antagonists. In SERT +/+ and +/- mice, the 5-HT1A auto receptor was primarily involved. However, in SERT -/- mice, the 5-HT7 receptor was the primary mediator of these temperature changes (Fox et al., 2008). As roles for 5-HT7 receptors in anxiety and depression were recently established and contributions of 5-HT1A receptors to therapeutic responses to serotonergic drugs have been well-established, the current findings have implications for understanding the high anxiety-like and depressive-like phenotype of SERT-deficient mice relevant to the treatment of human anxiety and affective disorders. In another series of studies based on case reports implicating the clinically-used atypical opioid, tramadol and another opioid that is an abused substance, meperidine, in the development of the human serotonin syndrome, we examined tramadol and meperidine as possible causative drugs in our SERT genetic mouse model. Comparisons were also made with SERT mice treated with morphine, an opioid not implicated in the serotonin syndrome in humans. We found that tramadol and meperidine, but not morphine, induced serotonin syndrome behaviors in mice, and that this response was exaggerated in SERT -/- and +/- mice (Fox et al., 2009). The exaggerated response to tramadol in SERT -/- mice was blocked by pretreatment with a serotonin receptor 5-HT1A antagonist. Further, we found that morphine-, meperidine- and tramadol-induced analgesia was markedly decreased in SERT -/- mice. These studies suggest that caution seems warranted in prescribing or not warning patients receiving SRIs or MAOIs that dangerous side-effects may occur during concurrent use of tramadol and similar agents. These findings in this mouse genetic model raise the possibility that humans with lower-expressing SLC6A4 SS genotypes or other SERT variants that lead to 50-80% decreases in SERT binding sites or transport function may be at higher risk to develop serotonin syndrome neurotoxicity. This is based on highly congruent data from imaging, neuroendocrine and other studies that have compared SERT-deficient mice to humans with SERT gene variants. Of special note, it is likely that relatively mild serotonin syndrome occurrence may contribute to early discontinuation of SRIs and other side effects during SRI treatment that are strongly associated with the lower-expressing SLC6A4 SS genotypes and S alleles as well as other newly discovered lower-expressing variants in this gene, as described in our other report, MH000336-30 LCS. (2). Another behavioral abnormality in SERT-deficient mice is reduced head twitch responses to serotonergic agents. This response is considered a pharmacologic proxy for hallucinogenic-like behaviors, as most of the drugs that induce this response in rodents are hallucinogens in humans, and these responses can be blocked by anti-psychotic drugs. In exploring mechanisms underlying this response, we found this year that reducing excess brain serotonin in SERT-deficient mice by using a serotonin synthesis inhibitor restored the deficient head twitch state in SERT -/- mice to levels of SERT control +/+ mice (Basselin et al., 2009). These findings provide a more comprehensive understanding of mechanisms in this genetic mouse model that eventually may provide insights into human disorders with genetic contributions that include hallucinogenic phenomena such as schizophrenia and bipolar disorders as well as the consequences of certain types of substance abuse disorders, all of major public health concern. (3). SERT-deficient mice were also compared to mice with gene-based deficiencies in other neurotransmitters, those for dopamine and norepinephrine, as well as to mice with other genetic deficiencies that interact with SERT, such as the integrin alphaIIbeta3 gene, as well as other types of environmental stimuli. Differences in brain, platelet and gut responses to multiple agents were observed. These findings serve as further illustrations of earlier data accumulated by our Lab that support the use of these mice as vulnerability models for humans with SERT gene variants with regard to gene-environment and gene-gene interactions that contribute to human diseases. (4). In other new studies of SERT-deficient mice, these mice were examined for possible autism-related traits, as some studies have identified associations of SLC6A4 polymorphisms as well as plasma serotonin levels with the occurrence of autism. In a three-chambered choice task, male SERT-deficient mice displayed significantly reduced social interaction behaviors with other mice compared to SERT +/+ mice (Moy et al., 2009). Thus, together with increased anxiety-like behaviors plus increased startle responses and increased behavioral and physiological responses to various types of stress, these mice show a series of features highly relevant to symptoms in several neuropsychiatric disorders of important public health concern.